Initial characterisation of adult human ovarian cell populations isolated by DDX4 expression and aldehyde dehydrogenase activity

Yvonne L Clarkson,Martin Waterfall,Paul A Skehel,Richard A Anderson,Evelyn E Telfer,Marie Mclaughlin,Cheryl E Dunlop

doi:10.1038/s41598-018-25116-1

Abstract

The existence of a population of putative stem cells with germline developmental potential (oogonial stem cells: OSCs) in the adult mammalian ovary has been marked by controversy over isolation methodology and potential for in-vitro transformation, particularly where cell sorting has been based on expression of DEAD box polypeptide 4 (DDX4). This study describes a refined tissue dissociation/fluorescence-activated cell sorting (FACS) protocol for the ovaries of adult women which results in increased cell viability and yield of putative OSCs. A FACS technique incorporating dual-detection of DDX4 with aldehyde dehydrogenase 1 (ALDH1) demonstrates the existence of two sub-populations of small DDX4-positive cells (approx. 7 µm diameter) with ALDH1 activity, distinguished by expression of differentially spliced DDX4 transcripts and of DAZL, a major regulator of germ cell differentiation. These may indicate stages of differentiation from a progenitor population and provide a likely explanation for the expression disparities reported previously. These findings provide a robust basis for the further characterisation of these cells, and exploration of their potential physiological roles and therapeutic application.

Highlights

The question of whether adult female mammals can undergo post-natal germ cell renewal has been debated for almost a century[1] with the current consensus being that the gamete pool is fixed before or around the time of birth, depending on species[2]
As an adjunct to sorting dissociated cell samples on this basis alone we hypothesised that the activity of a widely recognised marker of viable stem cells, aldehyde dehydrogenase 1 (ALDH1)[25], would be present in putative oogonial stem cells (OSCs)
There still remains little evidence to support neo-oogenesis under physiological conditions but there is an increasing body of work supporting the presence of cells within adult ovaries that may be oogonial stem cells (OSCs)

Summary

Introduction

The question of whether adult female mammals can undergo post-natal germ cell renewal has been debated for almost a century[1] with the current consensus being that the gamete pool is fixed before or around the time of birth, depending on species[2]. Since the isolation of mitotic cells expressing germline markers has been reported from ovaries of adult rodents[4,5,6,7,8,9], cows[10], sheep[11] primates[12] and humans[5,10,11,13,14,15,16] Development of these putative oogonial stem cells (OSCs) in-vitro[4,5,6,7,8,9,11,12,13,14,16] and the generation of live young from fully differentiated rodent putative OSCs6–9 has been described, no physiological role has been demonstrated in any species. Preliminary analysis of the ability of DDX4-positive sorted cells to develop into oocyte like structures when combined with somatic cells was performed

Methods

Results

Conclusion